Metastasis is a leading prognostic indicator for cancer survival and a major contributor to cancer mortality. The skeleton is one of the preferred organs for cancer dissemination in various malignancies. Current targeted cancer therapies are designed to alter not only specific biological functions in cancer cells, but also components of the tumor stroma/microenvironment such as blood vessels. Contrary to the established role of pericytes limiting cancer cell dissemination from the primary tumor, the role(s) of these stromal components at the target organ during cancer metastasis are not understood. The main objective of this research proposal is to understand the function of the Bone Marrow (BM) microenvironment, and specifically of its vascular components, in the establishment of skeletal metastasis, based on a novel hypothesis that suggests that BM Mesenchymal Stem Cells (BM-MSCs), as perivascular cells (pericytes), function as gatekeepers controlling cancer cell invasion to the bone. This new hypothesis provides clinically relevant information for therapeutic strategies that innovately aim at reducing the engraftment of circulating cancer cells by closing the gate through which the metastatic cell transit into the normal bone. To experimentally do so, a genetic model of perturbed pericyte coverage of the vasculature (PDGF-Bret/ret mice), in which the physical association between endothelial cells and pericytes is disrupted due to altered PDGF-B signaling, will be used to test the BM engraftment of circulating osteotropic melanoma and breast cancer cells. This model will help: first, to establish how the integrity/stability of te BM sinusoidal network, and the presence of BM-MSCs in their perivascular niche, participate in the extravasation of cancer cells (SA1); and second, to identify the cellular interactions and molecular pathways required for this cancer cell/tissue stroma relationship during BM invasion. These interactions will be further tested using in vitro and in vivo models (SA2). In SA1, Bioluminescence will be used to track the BM engraftment of circulating cells, their progression in time as metastatic tumors and the anatomical/functional correlation of resulting osteolysis (analyzed by ?CT imaging), which will be also assessed by histology, histomorphometry and serum markers. Immunohistochemical analysis using specific cellular markers will reveal the details of the interaction between engrafted melanoma cells and the altered vascular components of the BM stroma. In SA2 a mechanistic assessment will be performed using in vivo and in vitro assays with genetically modified MSCs, melanoma and breast cancer cells. In addition to the significant direct clinical impact, this proposed work is expected also to provide the foundation for future projects addressing other known roles of BM-MSCs that may influence skeletal metastasis, such as regulation of locally of other osteotropic malignancies including prostate, renal and lung, thus broadening the significance of the findings and conclusions. PUBLIC HEALTH RELEVANCE: The skeleton is one of the preferred organs for cancer dissemination and engraftment in various malignancies, negatively impacting patients' quality of life, therapeutic success and, thus, survival rates. We propose to understand the mechanism(s) behind the establishment of melanoma and breast cancer skeletal metastasis, based on a novel hypothesis which suggests that local adult stem cells in the bone marrow (BM- MSC), acting as pericytes, function as gatekeepers controlling cancer cell invasion to the bone, and preliminary results indicating a differential effect of those cells during intravasation (at the primary tumor) and extravasation (at the target organ). This new hypothesis provides clinically relevant information for therapeutic strategies that innovately aim at reducing the metastasis engraftment by closing the gate through which the metastatic cancer cell transit into the normal bone, thus improving the devastating outcomes.